Flexible and high-throughput sequencing of targeted genomic regions

Abstract

The disclosure pertains to materials and methods for capturing a target genomic region, comprising hybridizing an extension probe and a ligation probe to target sequences that flank the target genomic region; elongating the 3′ end of the extension probe until the 3′ end of the elongated extension probe is adjacent to the 5′ end of the ligation probe; and ligating the 3′ end of the elongated extension probe with the 5′ end of the ligation probe to produce a ligated probe. The ligated probe can be PCR amplified to produce copies of the target genomic region that can be detected or sequenced. Certain embodiments of the invention also provide methods of producing double stranded probes suitable for capturing and analyzing both strands of a target genomic region in a double stranded genomic DNA. The invention also provides kits for performing the methods disclosed herein.

We claim: 
     
         1 . A method of producing a double stranded oligonucleotide probe optionally having modifications on both strands at one end, the method comprising:
 a) providing a single stranded or double stranded pre-probe comprising from the 5′ end towards the 3′ end: a first tail, a first restriction site for a first restriction enzyme, a target binding sequence, a second restriction site for a second restriction enzyme, and a second tail, wherein the double stranded pre-probe is optionally produced in a PCR using appropriate primers to copy the single stranded pre-probe,   b) optionally, performing a tail-swap reaction to substitute a temporary first or second tail for a permanent tail that is genetically modified to comprise at least a portion of a new desired sequence comprising:
 i) digesting the double stranded pre-probe with the first restriction enzyme to remove the first pre-tail, or a portion of it, to produce an overhang, and 
 ii) ligating to the double stranded pre-probe digested with the first restriction enzyme to the permanent tail, the permanent tail molecule that contains genetic modifications and at least a portion of the permanent tail comprising an overhang that is complementary to the overhang of the digested double stranded pre-probe, 
   c) optionally, purifying the digested double stranded pre-probe ligated to the permanent tail,   d) producing a double stranded probe by digesting the double stranded pre-probe with the second restriction enzyme to remove the second tail and produce a blunt end or sticky end within the target binding sequence, and   e) optionally, purifying the double stranded probe.   
     
     
         2 . The method of  claim 1 , wherein the single stranded pre-probe comprises a barcode between the first restriction site and the target binding sequence and/or the target binding sequence and the second restriction site. 
     
     
         3 . The method of  claim 1 , wherein the first and the second restriction enzymes are Type IIS restriction enzymes that cleave a double stranded DNA away from its recognition site. 
     
     
         4 . The method of  claim 3 , wherein the Type IIS restriction enzymes are BsaI and Mly1. 
     
     
         5 . The method of  claim 1 , wherein the genetic modifications of the permanent tail confer exonuclease protection, incorporate detectable nucleotides or modified nucleotides. 
     
     
         6 . The method of  claim 1 , wherein the pre-probe comprises between 20 and 200 nucleotides. 
     
     
         7 . The method of  claim 6 , wherein the first tail is between 10 to 30 nucleotides, the target binding sequence is between 10 and 60 nucleotides, and the second tail is between 10 to 30 nucleotides. 
     
     
         8 . The method of  claim 1 , said method further comprising converting the double stranded probes to single stranded probes. 
     
     
         9 . The method of  claim 1 , said method comprising:
 a) providing a single stranded or double stranded pre-probe comprising from the 5′ end towards the 3′ end: a first tail, a first restriction site for a first restriction enzyme, a target binding sequence, a second restriction site for a second restriction enzyme, and a second tail, wherein the double stranded pre-probe is optionally produced in a PCR using appropriate primers to copy the single stranded pre-probe,   b) performing a tail-swap reaction to substitute a temporary first or second tail for a permanent tail that is genetically modified to comprise at least a portion of a new desired sequence comprising:   i) digesting the double stranded pre-probe with the first restriction enzyme to remove the first pre-tail, or a portion of it, to produce an overhang, and   ii) ligating to the double stranded pre-probe digested with the first restriction enzyme to the permanent tail, the permanent tail molecule that contains genetic modifications and at least a portion of the permanent tail comprising an overhang that is complementary to the overhang of the digested double stranded pre-probe,   c) purifying the digested double stranded pre-probe ligated to the permanent tail,   d) producing a double stranded probe by digesting the double stranded pre-probe with the second restriction enzyme to remove the second tail and produce a blunt end or sticky end within the target binding sequence, and   e) purifying the double stranded probe.   
     
     
         10 . A method of capturing a target genomic region from a double stranded target genetic material, the method comprising the steps of:
 a) providing a double stranded upstream probe and a double stranded downstream probe, wherein the double stranded upstream probe comprises:
 i) a first extension probe comprising toward the 3′ end a first target binding sequence and toward the 5′ end a first primer binding sequence, and 
 ii) a second ligation probe comprising toward the 5′ end a first target binding sequence and toward the 3′ end a first primer binding sequence, 
   and wherein the double stranded downstream probe comprises:
 i) a first ligation probe comprising toward the 5′ end a second target binding sequence and toward the 3′ end a second primer binding sequence, and 
 ii) a second extension probe comprising toward the 3′ end a second target binding sequence and toward the 5′ end a second primer binding sequence; 
   b) contacting the double stranded target genomic region with the double stranded upstream probe and the double stranded downstream probe, the contacting performed under conditions to allow:
 i) denaturation of the double stranded upstream probe and the double stranded downstream probe into the first extension probe, the second ligation probe, the first ligation probe and the second extension probe, and 
 ii) hybridization of the first extension probe and the first ligation probe to a first DNA strand in the target genomic region and hybridization of the second extension probe and the second ligation probe to a second DNA strand in the target genomic region, 
   c) amplifying the 3′ end of the first extension probe until the 3′ end of the amplified first extension probe is adjacent to the 5′ end of the first ligation probe and amplifying the 3′ end of the second extension probe until the 3′ end of the amplified second extension probe is adjacent to the 5′ end of the second ligation probe; and   d) capturing the target genomic region from the double stranded target genetic material by:
 i) ligating the 3′ end of the amplified first extension probe with the 5′ end of the first ligation probe to produce a first ligated probe, the first ligated probe comprising, from the 5′ end to the 3′ end, the first primer binding sequence, the first target binding sequence, the amplified target genomic region, the second target binding sequence, and the second primer binding sequence, and 
 ii) ligating the 3′ end of the amplified second extension probe with the 5′ end of the second ligation probe to produce a second ligated probe, the second ligated probe comprising, from the 5′ end to the 3′ end, the second primer binding sequence, the second target binding sequence, the amplified target genomic region, the first target binding sequence, and the first primer binding sequence. 
   
     
     
         11 . The method of  claim 10 , wherein the double stranded upstream probe comprises an exonuclease protection at the 5′ end of the first extension probe and an exonuclease protection at the 3′ end of the second ligation probe and the double stranded downstream probe comprises an exonuclease protection at the 3′ end of the first ligation probe and an exonuclease protection at the 5′ end of the second extension probe. 
     
     
         12 . The method of  claim 11 , wherein the exonuclease protection comprises one or more of: introducing thiophosphate linkages between nucleotides, incorporating two or more phosphoramidite, phosphoromonothioate and/or phosphorodithioate linkages, replacing one or more phosphodiester linkages between adjacent nucleotides by a formacetal/ketal linkage, blocking the 3′ terminal hydroxyl group by a phosphoryl or acetyl group, introducing 3′ terminal phosphoroamidate, introducing peptide nucleic acids (PNAs) or locked nucleic acids (LNAs), introducing one or more thiophosphate groups, or introducing a 2-O-methyl ribose sugar group in the oligonucleotide backbone. 
     
     
         13 . The method of  claim 10 , comprising isolating the first and the second ligated probes from the reaction mixture. 
     
     
         14 . The method of  claim 11 , wherein isolating the first and the second ligated probes comprises treating the reaction mixture with one or more exonucleases having a 5′-3′ exonuclease activity and a 3′-5′ exonuclease activity. 
     
     
         15 . The method of  claim 10 , wherein the target genomic region is between about 10 bp and about 100 bp, between about 100 bp and about 300 bp, between about 300 bp and about 1,000 bp or between about 1,000 bp and about 20,000 bp. 
     
     
         16 . The method of  claim 10 , further comprising amplifying the first and/or the second ligated probe in a polymerase chain reaction (PCR) using an amplification primer pair to produce copies of the first and/or the second ligated probe in a double stranded form,
 the first ligated probe amplification primer pair comprising:   i) a first extension probe amplification primer comprising from 5′ to the 3′ end, one or more of: a first sequencing primer binding sequence, a first identifier sequence, and the first primer sequence, and   ii) a first ligation probe amplification primer comprising from the 5′ to the 3′ end, one or more of: a second sequencing primer binding sequence, a second identifier sequence, and the second primer sequence; and   the second ligated probe amplification primer pair comprising:   i) a second extension probe amplification primer comprising from the 5′ to the 3′ end, one or more of: a third sequencing primer binding sequence, a third identifier sequence, and the second primer sequence, and   ii) a second ligation probe amplification primer comprising from the 5′ to the 3′ end, one or more of: a fourth sequencing primer binding sequence, a fourth identifier sequence, and the first primer sequence.   
     
     
         17 . The method of  claim 16 , further comprising sequencing the PCR amplified first ligated probe and/or the PCR amplified second ligated probe. 
     
     
         18 . The method of  claim 17 , wherein the sequencing comprises nanopore sequencing, reversible dye-terminator sequencing, Single Molecule Real-Time (SMRT) sequencing, or paired end sequencing. 
     
     
         19 . A kit comprising one or more pairs of double stranded probes, each pair of double stranded probe comprising a double stranded upstream probe and a double stranded downstream probe, wherein the double stranded upstream probe comprises:
 i) a first extension probe comprising toward the 3′ end a first target binding sequence and toward the 5′ end a first primer binding sequence, and   ii) a second ligation probe comprising toward the 5′ end a first target binding sequence and toward the 3′ end a first primer binding sequence,   and wherein the double stranded downstream probe comprises:   i) a first ligation probe comprising toward the 5′ end a second target binding sequence and toward the 3′ end a second primer binding sequence, and   ii) a second extension probe comprising toward the 3′ end a second target binding sequence and toward the 5′ end a second primer binding sequence.   
     
     
         20 . The kit of  claim 19 , wherein the double stranded extension probe comprises an exonuclease protection at the 5′ end of the first extension probe and an exonuclease protection at the 3′ end of the second ligation probe and the double stranded ligation probe comprises an exonuclease protection at the 3′ end of the first ligation probe and an exonuclease protection at the 5′ end of the second extension probe. 
     
     
         21 . The kit of  claim 20 , wherein the exonuclease protection comprises one or more of: thiophosphate linkages between nucleotides, two or more phosphoramidite and phosphoromonothioate and/or phosphorodithioate linkages, one or more phosphodiester linkages between adjacent nucleotides by a formacetal/ketal linkage, blocked 3′ terminal hydroxyl group by a phosphoryl or acetyl group, 3′ terminal phosphoroamidate, peptide nucleic acids (PNAs) or locked nucleic acids (LNAs), one or more thiophosphate groups, or a 2-O-methyl ribose sugar group in the oligonucleotide backbone.